Next Generation Sequencing (NGS)

Learn about Next Generation Sequencing using Illumina MiSeq™ Instrumentation at the Massey Genome Service – including detailed submission process information and specifications.

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Illumina MiSeq™ instrument.

Next generation sequencing services overview

Massey Genome Service (MGS) has invested in two Illumina MiSeq™ instruments. We offer Illumina MiSeq™ sequencing with the following applications:

  • Genomic DNA sequencing of small genomes such as bacterial, fungal and viral
    de novo sequencing and re-sequencing
  • Whole Metagenomics
  • 16S amplicon Sequencing
  • Custom amplicon sequencing
  • RNA sequencing: Gene Expression analysis, Transcriptomics and
    Small RNA Sequencing

Massey Genome Service is an agent for HiSeq, Novaseq, PacBio sequencing services.

Submit an email enquiry

Send the Massey Genome Service (MSG) an email about your project at: mgs-manager@massey.ac.nz.

Giving us an outline of your project requirements means we can make informed decisions about your project design and requirements.

Include the following information in your email:

  • Client’s Name
  • PI Name
  • Client’s Organisation/Institute Name
  • Organisation/Institute paying for the Project (if different from Organisation/Institute)
  • Client’s phone number
  • Client’s email address
  • Additional client’s/contacts
  • Person who will be downloading the data when the project is completed
  • Project Title
  • Description of Project and Objectives – Aim of the project: What are the scientific aims of the project, and the desired outcomes? What do you want the data to tell you?
  • Request for project advise on: Sample requirements, platform choice, Data analysis
  • Experimental Plan (if known): Please describe the basic experimental plan including sample number, library type and number/type of Illumina MiSeq™ runs
  • Bioinformatics analysis required (Yes/No)
  • Do you require the Express Service or the Standard Service?

Express service

Priority is placed on providing a faster turnaround time for the project.

Data will be delivered to the client within 1 to 2 weeks, depending on the size of the project, from the date of delivery of the samples to the service.

There is an additional 30% premium on the cost associated with the Express Service and this additional cost is stated in the Project Plan/Quotation for each project.

Standard service

There is no priority placed on providing a fast turnaround time for the project. Data will be delivered to the client within 5-8 weeks, depending on the size of the project, from the date of delivery of the samples to the service.

You will receive an e-mail response back from the MGS Service Team within 2 working days. The service team may ask for more information to be supplied and a decision will be made on the best approach to take for your project.

Project plan and quotation

We will put together a project plan and quotation and email it to you for review. To proceed with the project sign the Project Plan/Quotation and email it back to us at mgs-manager@massey.ac.nz. Include an electronic copy of a Purchase Order (PO) for payment.

You will need to raise the PO with your organisation's accounts department. For external clients (projects not funded by Massey) the project plan and quotation will be accompanied by a service contract. This needs to be signed, dated and an electronic copy returned to the MGS Service Team at mgs-manager@massey.ac.nz.

Sample preparation and sample quality data requirements

After accepting the project plan and quotation (and service contract for external clients) you will be asked to prepare your samples, and send us data about their quality.

We will assess the quality of your samples before you send them to the service. This is a preliminary check of sample quality. The service will also carry out sample QC checks when the samples arrive at the facility.

Do not send your samples to us until you have been notified to do so. If your quality data does not meet the standards for sample quality you will be informed on procedures to take to improve the quality of your samples.

Detailed sample preparation, quantification and quality requirements

Get detailed requirements for preparing libraries and samples.

Illumina MiSeq™ sequencing applications

Table of Illumina MiSeq™ sequencing applications.
Application Type of Sample to Supply Illumina MiSeq™ Read lengths recommendations Method of Library Preparation Minimum Amount Required Minimum Concentration Required Buffer
Whole Genomic DNA Sequencing (WGS) of small genomes (De novo assembly) Fungal, bacterial, viral and smaller complex genomes Illumina MiSeq™ V2 (2x 250 base PE)* or Illumina MiSeq™ V3 (2x 300 base PE)! Illumina TruSeq™ DNA PCR-Free Library Preparation 1.5µg DNA (350 bp insert) or 2.5µg DNA (550 bp insert) 20ng/µl 10mM Tris-HCl, pH8.5 or molecular grade water
Whole Genomic DNA Sequencing of small genomes (Re-sequencing) Fungal, bacterial, viral and smaller complex genomes Illumina MiSeq™ V2 (2x 250 base PE)* or Illumina MiSeq™ V3 (2x 300 base PE)! Illumina TruSeq™ DNA PCR-Free Library Preparation 1.5µg DNA (350 bp insert) or 2.5µg DNA (550 bp insert) 20ng/µl 10mM Tris-HCl, pH8.5 or molecular grade water
Whole Genomic DNA Sequencing of small genomes (Re-sequencing) Illumina TruSeq™ DNA Nano Library Preparation 1.5µg DNA (350 bp insert) or 2.5µg DNA (550 bp insert) 20ng/µl
Whole Genomic DNA Sequencing of small genomes (Re-sequencing) Illumina Nextera™ DNA Library Preparation 100ng DNA 10ng/µl
Whole Genomic DNA Sequencing of small genomes (Re-sequencing) Illumina Nextera XT™ DNA Library Preparation 1.5ng DNA 0.2ng/ µl
Whole Metagenomic Sequencing Microbial environmental samples containing a population of microbes Illumina MiSeq™ V2 (2x 250 base PE)* or Illumina MiSeq™ V3 (2x 300 base PE)! Illumina TruSeq™ DNA PCR-Free Library Preparation 1.5µg DNA (350 bp insert) or 2.5µg DNA (550 bp insert) 20ng/µl 10mM Tris-HCl, pH8.5 or molecular grade water
Illumina TruSeq™ DNA Nano Library Preparation 1.5µg DNA (350 bp insert) or 2.5µg DNA (550 bp insert) 20ng/µl
Illumina Nextera™ DNA Library Preparation 100ng DNA 10ng/µl
Illumina Nextera XT™ DNA Library Preparation 1.5ng DNA 0.2ng/ µl
PCR Amplicon Sequencing Large Amplicons ≥ 2Kb Illumina MiSeq™ V2 (2x 250 base PE)* or Illumina MiSeq™ V3 (2x 300 base PE)! Illumina Nextera™ DNA Library Preparation 100ng DNA 10ng/µl 10mM Tris-HCl, pH8.5 or molecular grade water
Illumina Nextera XT™ DNA Library Preparation 1.5ng DNA 0.2ng/ µl
Custom Amplicon Sequencing Amplicons ≤ 550bp Illumina MiSeq™ V2 (2x 150 base PE)* or Illumina MiSeq™ V2 (2x 250 base PE)* or Illumina MiSeq™ V3 (2x 300 base PE)! (Run length is dependent on the size of the amplicons being sequenced) Illumina Two Step PCR Amplicon Approach using the Illumina Nextera XT ™ DNA Primers for the second PCR step. Client is responsible for carrying out the first PCR step using custom made Illumina tailed primers (Additional information is provided upon scoping of the project) 25µL of the amplicon generated from the first PCR step ≥ 1.0ng/µl 10mM Tris-HCl, pH8.5 or molecular grade water
Metagenomic 16S Amplicon Sequencing V3-V4 hypervariable region Genomic DNA or PCR Amplicon containing the 16S gene (Amplicon size = ~459 bp) Illumina MiSeq™ V2 (2x 250 base PE)* 5-50ng DNA 5ng/µl 10mM Tris-HCl, pH8.5 or molecular grade water
RNA Sequencing Gene Expression Analysis (Transcriptomics) Total RNA Illumina MiSeq™ V2 (2x 150 base PE)* or Illumina MiSeq™ V3 (2x 75 base PE)! Illumina TruSeq™ Stranded Total RNA Library Preparation 1.5µg Total RNA 200ng/µl 10mM Tris-HCl, pH8.5 or molecular grade water
Illumina TruSeq™ Stranded mRNA Library Preparation 1.5 µg Total RNA 200ng/µl

* Version 2 Illumina MiSeq™ chemistry. Refer to “Illumina MiSeq™ Run Options” for details.
! Version 3 Illumina MiSeq™ chemistry. Refer to “Illumina MiSeq™ Run Options” for details.
# Reference: http://aem.asm.org/content/79/17/5112

Do not use buffer containing EDTA

Do not send the samples in a buffer which contains EDTA when preparing samples ready for the Illumina Nextera™ DNA Library preparation or Illumina Nextera™ XT DNA Library preparation.

EDTA is known to be an inhibitor in enzymatic reactions and could inhibit the enzymatic fragmentation used in the Illumina Nextera ™ DNA Library preparation and Illumina Nextera™ XT DNA Library preparation methods.

Minimum product size

The minimum product size for the Illumina Nextera™ XT DNA Library preparation is 300 bp. This is to ensure even coverage across the length of the DNA fragment.

An expected drop off in sequencing coverage about 50 bp from each distal end of a fragment may be seen. This is because the transposon tagmentation enzymatic reaction used to randomly fragment the product cannot add an adapter right at the distal end of a fragment. This enzymatic clipping of PCR primers avoids wasted sequencing output on non-informative bases that do not contain genomic inserts.

If you wish to sequence the genomic loci contained within a PCR primer, simply design your amplicons to be ~100 bases larger than the desired insert to be sequenced.

Preparation of DNZ samples

We recommend you use column based purification methods to extract the genomic DNA. DNA samples should be treated with RNase prior to purification on the column.

Drying the column after the wash step with a second spin is essential to remove excess ethanol, which will interfere with library preparation. Avoid the use of organic extraction methods, such as phenol or Trizol, which can interfere with the enzymatic reactions used during the library preparation protocols.

Quantitate and assess quality

You must quantitate the Genomic DNA or PCR Amplicon and assess the quality of the DNA before delivering samples for library preparation and Illumina MiSeq™ sequencing.

~150ng of genomic DNA samples must be run on a 0.5-1% agarose gel with a high molecular weight ladder. MGS will NOT run a gel when the samples arrive at the service.

PCR Amplicons must be run on either a 1-2% agarose gel with a ladder containing similar band sizes to your products, or bioanalyzer such as Agilent 2100 Bioanalyzer, if the client has access to this instrumentation, using either the Agilent DNA 7500 or Agilent DNA 12000 Labchip Kit.

LabChip service

As an alternative, we provide a LabChip Service. Clients can provide Genomic DNA or PCR Amplicons and the Massey Genome Service will run them using the PerkinElmer GX Touch HT Instrument using:

  • DNA High Sensitivity Assay – PCR Amplicons
  • Genomic DNA Assay – Genomic DNA

What you must supply

You must supply an electronic copy of the 1-2% agarose gel image, or the summary report from the bioanalyzer, and the fluorometer such as Qubit concentration readings and spectrophotometer OD260/280 ratio and OD260/230 ratio readings.

Quality standards

High quality genomic DNA should run as a high molecular weight band on a 1-2% agarose gel, with the majority of DNA greater than 10Kb in size and with minimal lower molecular weight smearing.

If the majority of the DNA is below 10Kb or smearing is visible, this suggests that the DNA is degraded or lower molecular smearing can be indicative of the presence of RNA contamination. The MGS will not accept your samples for sequencing if visible smearing is seen.

The spectrophotometer OD260/280 ratio should be 1.8-2.0; OD260/230 ratio should be 2.0-2.2

Preparation of RNA Samples

Total RNA should be extracted using a column purification method. Organic extraction methods, such as phenol or Trizol, are discouraged for the purification of total RNA.

Tissues with a high fat content and certain embryonic tissues require an organic step to remove the excess amount of these other bio-molecules. For this we recommend the use of one of the following Qiagen kits:

  • Qiagen RNeasy Lipid Tissue Mini Kit
  • Qiagen RNeasy microarray tissue mini kit
  • Qiagen miRNeasy mini kit.

All three of these kits include an organic step prior to column purification. Residual DNA can also be removed during the purification process when using any of these three kits.

Run samples on a bioanalyzer

Total RNA samples must be run on a bioanalyzer such as Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Nano Labchip Kit, if the client has access to this instrumentation.

Alternatively run the total RNA on a formaldehyde 1% agarose gel with an RNA 6000 ladder and measure the integrity of the RNA upon staining with ethidium bromide.

LabChip service

As an alternative, we provide a LabChip Service. For the LabChip Service clients can provide Total RNA or mRNA to the MGS and the service will run them using the PerkinElmer GX Touch HT Instrument using the Standard RNA Assay.

High-quality RNA

It is very important to use high-quality RNA as the starting material. The use of degraded RNA can result in a low yield, over-representation of the 3’ ends of the RNA molecules, or failure of the library preparation.

A DNase step must be included as part of the RNA isolation method. The presence of DNA contamination may result in an underestimation of the amount of RNA used.

What you must supply

The client must supply us with an electronic copy of the formaldehyde agarose gel image, or summary report from the bioanalyzer, and the fluorometer such as Qubit concentration readings and spectrophotometer OD260/280 ratio and OD260/230 ratio readings.

Quality standards

Bioanalyzer Trace

An RIN or RQS value greater than 8 is required, or greater than 7 for plant RNA and rRNA ratio (28S/18S or 23S/16S) of 1.5 to 2.5.

Formaldehyde 1% Agarose

High quality RNA shows a 28S rRNA band at 4.5Kb that should be twice the intensity of the 18S rRNA band at 1.9Kb. Both kb determinations are relative to a RNA 6000 ladder. mRNA will appear as a smear from 0.5-12Kb.

The spectrophotometer OD260/280 ratio should be 1.8-2.2; OD260/230 ratio should be 2.0-2.2.

Client-prepared libraries

We accept client-prepared libraries using Illumina compatible library preparation methods. However, an MGS disclaimer will be included in the MGS Project Plan, as we are not able to guarantee the quality or quantity of data generated from client prepared libraries.

Client-prepared libraries must be run on a bioanalyzer such as Agilent 2100 Bioanalyzer, if the client has access to this instrumentation, using either the Agilent DNA 1000 or Agilent DNA High Sensitivity Labchip Kit. If you do not have access to a bioanalyzer then we will QC check the libraries upon arrival at our facility.

You must supply us with the summary report from the bioanalyzer.

Quality standards for libraries prepared using Illumina Kits

High quality libraries should have a clean single peak as follows with no noticeable adapter dimer or PCR primer peaks.

~470-670 bp for libraries prepared using the Illumina TruSeq™ DNA PCR-Free or Illumina TruSeq™ DNA Nano Library Preparation methods.

~250-350 bp for libraries prepared using the Illumina TruSeq™ RNA or Illumina TruSeq™ Stranded Total RNA Library Preparation methods.

~175-700 bp for libraries prepared using the Illumina Nextera™ or Illumina Nextera™ XT Library Preparation methods.

~570-600 bp for libraries prepared for 16S amplicon sequencing of the V3-V4 rRNA hyper-variable region.

Library preparation methods

Below is a list of the library preparation methods used by the Massey Genome Service to prepare libraries for the Illumina MiSeq™ and Illumina HiSeq™ instruments.

Illumina TruSeq™ DNA PCR-Free Library Preparation

This method uses mechanical shearing to randomly fragment the genomic DNA. Magnetic bead size selection will be used to select fragments with an average size of 350bp or 550bp and then barcoded Illumina adapters will be ligated onto each end of the fragments. This library preparation method does not include a PCR enrichment step.

This method is suitable for preparing libraries from genomic DNA for whole genome sequencing (De novo assembly or re-sequencing) and whole metagenomic sequencing.

Illumina TruSeq™ DNA Nano Library Preparation

This method uses mechanical shearing to randomly fragment the genomic DNA. Magnetic bead size selection will be used to select fragments with an average size of 350bp or 550b and then barcoded Illumina adapters will be ligated onto each end of the fragments. The library products are then enriched for products which are fully ligated.

This method is suitable for preparing libraries from genomic DNA for whole genome sequencing (Re-sequencing) and whole metagenomic sequencing.

Illumina Nextera™ DNA Library Preparation

This method use enzymatic shearing to randomly fragment the genomic DNA. Barcoded Illumina adapters will be added onto each end of the fragments during enrichment PCR.

Do not send the samples in a buffer which contains EDTA when preparing samples for the Illumina Nextera™ DNA Library preparation. EDTA is known to be an inhibitor in enzymatic reactions and could inhibit the enzymatic fragmentation used in the Illumina Nextera ™ DNA Library preparation method.

This method is suitable for preparing libraries from genomic DNA for whole genome sequencing (Re-sequencing), whole metagenomic sequencing and PCR amplicon sequencing (amplicon ≥ 2Kb).

Illumina Nextera™ XT DNA Library Preparation

This method use enzymatic shearing to randomly fragment the genomic DNA. Barcoded Illumina adapters will be added onto each end of the fragments during enrichment PCR.

Do not send the samples in a buffer which contains EDTA when preparing samples for the Illumina Nextera™ DNA Library preparation. EDTA is known to be an inhibitor in enzymatic reactions and could inhibit the enzymatic fragmentation used in the Illumina Nextera ™ XT DNA Library preparation method.

This method is suitable for preparing libraries from genomic DNA for whole genome sequencing (Re-sequencing) of small genomes, whole metagenomic sequencing and PCR amplicon sequencing (amplicon ≥ 2Kb).

Illumina TruSeq™ RNA

This method generates mRNA targeted libraries from Total RNA, and uses oligo dT beads to extract poly-adenylated mRNA from total RNA. RNA must be of high integrity so as to extract full length transcripts, and minimise 3’ bias. Does not provide strand information on RNA transcripts.

This method is suitable for preparing libraries from total RNA for RNA sequencing, gene expression analysis and transcriptomics.

Illumina TruSeq™ Stranded Total RNA

This method captures both coding RNA, as well as multiple forms of non-coding RNA. Strand information on RNA transcripts is obtained.

This method is suitable for a range of samples including low-quality and FFPE. It employs Ribo-Zero ribosomal reduction chemistry to eliminate the ribosomal fractions. The method works best on human, rat and mouse samples, but can be used less optimally on other samples.

Information for rRNA Depletion using Illumina Ribo-Zero Plus

This method is suitable for preparing libraries from total RNA for RNA sequencing, gene expression analysis and transcriptomics.

16S V4 Amplicon

The library is prepared using a single PCR amplification from genomic DNA samples, using Illumina tailed single indexed primers which bind to the regions flanking the V4 hyper-variable region of 16S rRNA.

Up to 48 samples can be multiplexed onto one Illumina MiSeq run. In most microbial species, the V4 hyper-variable region of 16S rRNA is approximately 254 bp, and only deviates from this length by a few base pairs. With the Illumina sequencing both strands of this 254 bp region are sequenced using a 150 bp read.

16S V3-V4 Amplicon

The library is prepared using a single PCR amplification from genomic DNA samples, using Illumina tailed single indexed primers which bind to the regions flanking the V3-V4 hyper-variable region of 16S rRNA.

Up to 384 samples can be multiplexed onto one Illumina MiSeq run. In most microbial species, the V3-V4 hyper-variable region of 16S rRNA is approximately 460-470 bp in length. With the Illumina sequencing both strands of this 460-470 bp region are sequenced using a either a 250 bp or 300 bp read.

Custom Amplicon

Custom amplicon libraries are prepared using “two step PCR” amplification from genomic DNA samples. The first PCR step amplified the target region of interest, using Illumina tailed primers containing a sequence which is complementary and will bind to the second step PCR primers.

The first step PCR primers should be designed to target a region of ≤ 550 bp. The second PCR step uses the Illumina Nextera dual index primers for PCR amplification, allowing up to 96 samples to be pooled onto one Illumina MiSeq run.

With the Illumina sequencing both strands of this ≤550 bp region are sequenced using a either a 250 bp or 300 bp read, depending on the size of the amplicon product.

Sample delivery

You will be asked via e-mail to send your samples by courier to us on dry ice for RNA samples or ice packs for DNA samples.

If you are local to the service you are welcome to hand deliver your samples. You will be emailed a copy of the Sample Submission Form. You must complete this and:

Instructions on sending samples

Get detailed instructions for sending samples.

Samples are to be delivered to the Massey Genome Service by courier at the clients own expense. Within New Zealand samples are to be delivered by domestic overnight courier service for express next day delivery, and international deliveries are by international courier services arranged by the client.

Samples are to be delivered on dry ice in liquid form for RNA samples or on ice packs in liquid form for DNA samples. If international deliveries are made on dry ice then it is a requirement that the dry ice is replenished during shipment to prevent thawing of samples.

Address to send samples to

Samples must be delivered to the following address for Illumina MiSeq™ sequencing:

Massey University
School of Natural Sciences (SNS)
Inward Goods
Science Tower A1.19
Columbo Road
Palmerston North 4410
ATTN: Massey Genome Service

Samples may be hand delivered to the service if you are situated locally and are able to do so.

Sample containers

Samples must be put into either 0.5mL or 1.5mL screw cap or flip lid microtubes, wrapped with parafilm. Label the tube(s) with the client party contact, NZGL contract number, sample name, total amount of sample (µg) delivered and volume (µl) if in liquid form.

Sample tubes must be put into a 50mL falcon tube(s) and packed with clean tissue for safe delivery to prevent/minimize possible breakages. Seal the 50mL falcon tube(s) with parafilm. If samples are being sent in dry form the falcon tube(s) must also be bubble wrapped.

International deliveries

International customers sending DNA or RNA samples for sequencing must send them with the Import Permit for importing materials of a biological nature into New Zealand.

The permit must be attached securely to the outside of the parcel so that it is available for customs inspection, along with written details as to the content of the parcel.

Write the following note to be sent with the import permit: "Purified DNA (or RNA), non-hazardous, non-infectious, and non-viable."

New Zealand import regulations

New Zealand has very strict import regulations, and the import permit must be present with the sample(s) for customs inspection.

The biological import permit is updated regularly, so each time the client comes to send samples please e-mail x.x.lin@massey.ac.nz for Illumina MiSeq™ sequencing to ask for the most recent permit.

Once courier arrangements have been made, email us tracking information along with the following information:

  • client’s organisation name, address and your contact details.
  • Name of the courier company the client is using and their contact details.
  • Date the parcel was sent.

Total amount and concentration of samples to supply can be found in the previous section.

Sample quality assessment and report

When the samples arrive at the Massey Genome Service, a quality and quantification assessment will be carried out on the samples, before the service proceeds with the library preparation.

If the samples, client-prepared libraries or service-prepared libraries do not meet our quality or quantification standards, you will be required to sign our disclaimer. Email the signed disclaimer to mgs-manager@massey.ac.nz if you wish to continue with the processing of the samples and/or libraries.

You will incur the cost of the sample quality assessment whether or not you decide to continue. Should your samples meet the services quality standard, then the service will continue with the library preparation.

Sample and library QC checks performed by the service

See details on quality and quantification assessment.

MGS will run client samples on the Qubit® Fluorometer or PerkinElmer Victor Plate Reader using the Quant-IT dsDNA HS Assay, as a quantification check before library preparation.

The service will also run client and service prepared libraries on the Qubit® Fluorometer or PerkinElmer Victor Plate Reader, and the PerkinElmer LabChip® GX Touch HT instrument, as a quality control check before sequencing on the Illumina MiSeq™ instrument.

Qubit® Fluorometer and PerkinElmer Victor Plate Reader

The following Invitrogen® assays are run:

  • Quant-iT™ HS DNA Assay or Quant-iT BR DNA Assay – For genomics DNA and PCR amplicon samples, and MGS and client prepared libraries
  • Quant-iT™ RNA Assay – For Total RNA and mRNA samples

Note the Quant-iT™ HS DNA Assay is run to determine the percentage of DNA contamination for RNA samples. % contamination should be ≤ 10%.

PerkinElmer GX Touch HT Instrument

The following PerkinElmer LabChip® assays are run:

  • High Sensitivity DNA Assay – MGS and client prepared libraries.

If the samples, client-prepared libraries or service-prepared libraries do not meet our quality or quantification standards, you will be required to sign an MGS disclaimer should you wish to continue with the processing of the samples and/or libraries.

Summary of QC Requirements

Table summarising QC requirements
Sample type Customer requirements MGS services QC checks
Genomic, Metagenomic and PCR Amplicon samples - 1-2% Agarose gel- Spectrophotometer or Fluorometer to measure concentration and Spectrophotometer to measure OD260/280 and OD230/260 ratios - Qubit® Fluorometer or PerkinElmer Victor Plate Reader
Total RNA and Small RNA samples# - Bioanalyzer (if you have access)- Formaldehyde 1% Agarose gel (if you do not have access to Bioanalyzer)- Fluorometer to measure concentration and Spectrophotometer to measure OD260/280 and OD230/260 ratios - Qubit® Fluorometer or PerkinElmer Victor Plate Reader
Libraries - Service will accept prepared libraries from clients however an MGS disclaimer will be included in the Project Plan which the client needs to sign before proceeding with the Project - PerkinElmer GX Touch HT instrument using LabChip® DNA High Sensitivity Assay- Qubit® Fluorometer or PerkinElmer Victor Plate Reader

# We provide a LabChip Service.

Library preparation

We will proceed with the preparation of your libraries. Once the libraries have been prepared, a quality and quantification assessment will be carried out on the libraries. Then we will proceed with sequencing.

If the libraries do not meet our quality standards you will be sent a Library Quality Report containing a disclaimer, which will state why your libraries do not meet our quality standards.

This document must be signed and returned to MGS by mail at mgs-manager@massey.ac.nz authorising the service to either continue or not to continue with the sequencing of the libraries.

You will incur the cost of library preparation whether or not you decide to continue. Should your libraries meet the services quality standard, then the service will continue with the library preparation.

Sequencing of libraries

We will carry out the sequencing of the libraries once you send authorisation to the Massey Genome Service to do so.

The run quality is monitored in real-time, which monitors:

  • signal intensities
  • base quality scores
  • fluidics
  • imaging.

Analysis is performed during the run to save on downstream analysis time. Primary analysis generates quality-scored base calls from the raw image files, which contain base calls per cycle.

The detailed sample preparation, quantification and quality requirements section above includes Illumina MiSeq instrument run specifications and quality standards.

Data delivery

Data is delivered to you in a hard drive by overnight courier service along with a Sequencing Run Report and Data Quality Report containing:

  • sample quality information
  • library quality information
  • Illumina MiSeq™ run quality
  • data quality information.

The data is delivered in fastq format and the sequencing reads are quality trimmed to 0.01 probability.

Please note, data will not be delivered to customers until we have received a PO for payment.

Applications

The Illumina MiSeq™ sequencing through Massey University currently offers the following applications as a part of the services being offered.

Genomic DNA sequencing of small genomes e.g. bacterial, fungal and viral

Microbial sequencing targets single microbes in contrast with metagenomics, with the aim to discover of genetic variations.

de novo sequencing

DNA sequencing and assembly of novel genomes.

Re-sequencing

DNA sequencing and mapping to a reference to check for gene variations.

Whole Metagenomic sequencing

Metagenomics is the study of genetic material isolated from microbial communities.

This technical advancement has initiated the trend of sequencing multiple samples in different conditions or environments to explore the similarities and dissimilarities of the microbial communities.

16S Metagenomic sequencing

Metagenomic studies can be carried out using the prokaryotic 16S ribosomal RNA (rRNA) gene. The 16S rRNA gene is ~1500 bp and contains 9 hyper-variable regions, which can be sequenced for phylogenetic classification.

The protocol for 16S rRNA sequencing targets the V3 and V4 hyper-variable regions, generating an amplicon of ~459 bp, although the length will vary depending on the organisms. This methodology can be adapted and used to target various regions of the 16S gene, or any other gene of interest.

Custom amplicon sequencing

Custom amplicon sequencing or targeted DNA sequencing allows the researcher to utilize the specificity of PCR in order to target the genes of their choosing.

Targeted DNA sequencing utilizes the ability to generate deep sequencing coverage of a target gene or gene region, to identify genes expressed at lower levels that may possibly have been missed by other sequencing methods.

RNA Sequencing

  • Gene Expression analysis
  • Transcriptomics
  • Small RNA Sequencing

Get more information from the Illumina website

Storage and retention of samples

Storage of samples

All samples and client prepared libraries upon entering the facility are unpackaged and checked for breakages. If tubes are broken, then the client will be contacted immediately and will be asked to resend the samples or client prepared libraries.

All DNA samples and prepared libraries are stored in a -20oC freezer, and all RNA samples are stored in a -80oC chest freezer.

Retention and return of samples

All samples that enter the facility, both DNA and RNA, and prepared libraries are stored for a period of 6 months after project completion, after which samples and libraries could be destroyed.

During the 6 months after project completion, customers can request return of the samples. Please contact MGS lab manager (+64 6 951 9080). Libraries prepared by the service will not be returned to clients.

Data storage and delivery

Storage of data

All data is stored on the Illumina BaseSpace server for 3 months, which excludes image files.

All data is stored locally in the Illumina MiSeq™ instruments, which includes images for a period of 1-2 months. All data is backed up to hard drives, including images, for storage up to 6 month.

Delivery of data

The raw and quality checked data in fastq format, a “Sequencing Run Report” and “Data Quality Report” will be sent to clients on completion of the Project, subject to the terms and conditions of the MGS Project Plan. MGS will notify you when the data ready.

Illumina MiSeq Run Options

Table showing Illumina MiSeq Run Options.
Illumina MiSeq Platform Illumina MiSeq V2(2x 250 base PE)1 Illumina MiSeq V2(2x 150 base PE) 1 Illumina MiSeq V2(2x 25 base PE) 1 Illumina MiSeq Micro V2(2x 150 base PE) 1 Illumina MiSeq Nano V2(2x 150 base PE) 1 Illumina MiSeq Nano V2(2x 250 base PE) 1 Illumina MiSeq V3(2x 75 base PE) 2 Illumina MiSeq V3(2x 300 base PE) 2
Output ~6-7.5 Gb per run ~3-4.5Gb per run ~750-850Mb per run ~1.0-1.2Gb per run ~250-300Mb per run ~450-500Mb per run ~3-3.8Gb per run ~12-15Gb per run
Number of reads ~24-30 million paired-end reads per run ~24-30 million paired-end reads per run ~24-30 million paired-end reads per run ~ 6.7-8 million paired-end reads per run ~ 1.6-2 million paired-end reads per run ~1.6-2 million paired-end reads per run ~44-50 million paired-end reads per run ~44-50 million paired-end reads per run
MiSeq instrument Data Quality3 75% bases higher than Q30 at 2X250 bp 80% bases higher than Q30 at 2X150 bp 90% bases higher than Q30 at 2X25 bp 80% bases higher than Q30 at 2X150 bp 80% bases higher than Q30 at 2X150 bp 75% bases higher than Q30 at 2X250 bp 85% bases higher than Q30 at 2X75 bp 70% bases higher than Q30 at 2X300 bp

1. Version 2 Illumina MiSeq chemistry.
2. Version 3 Illumina MiSeq chemistry.
3. Illumina Quality Specifications.

The Illumina Micro and Nano Illumina Miseq kits are suitable for genomic sequencing projects where there are only a small number of small genomes to be sequenced, and also for small scale custom amplicon sequencing projects that require less reads.

See the Detailed sample preparation, quantification and quality requirements above to understand which run lengths are suitable for each application.

Contact us

All enquires and requests for NGS work must be made by contacting Xiaoxiao Lin, Laboratory/QA Manager.

E-mail: mgs-manager@massey.ac.nz

Phone: +64 6 951 9080 ext 86080

Please download the PDF file below, which provides the information for clients to follow from submission of an enquiry via email, through to delivery of the data.

Xiaoxiao Lin

Laboratory/QA Manager

Massey Genome Service

Office phone

+64 6 951 9080 (external)

Extension 86080 (internal customers)

Laboratory phone

+64 6 951 8735 (external)

Extension 85735 (internal customers)

Location

Physical address
Massey Genome Service
School of Natural Sciences
Room ScD3.15A, Riddet Road
Massey University
Turitea Campus
Palmerston North 4410
New Zealand

Postal address
Massey Genome Service
Massey University
Private Bag 11-222
Palmerston North 4442
New Zealand

Courier address
Massey Genome Service
School of Natural Sciences
Inward Goods
Science Tower A, Level 1,
Columbo Road, Massey University
Palmerston North 4410
New Zealand